Pulsed electron paramagnetic resonance studies of the copper complexes of transferrin.

The nuclear modulation effect in pulsed EPR spectroscopy was used to probe the metal-protein bonds in Cu(II)=transferrin complexes as a function of pH. In preparations in equilibrium with air at pH 4.8 no modulation features other than those attributable to protons were observed, in keeping with conventional EPR studies indicating that at this pH only nonspecific binding takes place (Zweier, J. L., and Aisen, P. (1977) J. Biol. Chem. 252,6090-6096). At pH 7.7, however, where each of the two specific sites of transferrin accommodates Cu(I1) with carbonate as the associated anion, a modulation pattern is obtained which indicates that an imidazole ligand is coordinated to specifically bound Cu(I1). This is consistent with the nitrogen superhyperfine structure in the EPR spectrum of Cu(II)* transferrinecarbonate, and corroborates chemical modification studies implicating histidine residues at the specific sites (Line, W. F., Grohlich, D., and Bezkorovainy, A. (1967) Biochemistry 6, 3393-3402). At pH 11.4, where the EPR spectrum shows three or four nitrogen ligands interacting with the first Cu(I1) bound to transferrin in the presence or absence of carbonate or other binding anions, the modulation envelope continues to indicate that imidazole is a ligand to the Cu(II), suggesting that the metal is bound to a specific site and not to a biurettype complex. A comparison of the envelopes of electron spin echoes of Cu(I1) l transferrin l [“C]oxalate and Cu(I1) l transferrin l [%]oxalate reveals the presence of a Cu(II)-13C superhyperfine interaction in the latter complex. This exceeds the free precession frequency of 13C by a factor greater than 2 and is indicative of a contact interaction between Cu(I1) and 13C. These results confirm that the anion is directly coordinated to the metal ion in the specific Cu(I1) l transfer& l oxalate complex.